The long-term goal of the proposed project is to develop a continuous and genetically encoded source of new antibiotics against drug-resistant bacterial pathogens by combining the power of recombinant DNA technology with the biosynthesis of natural product antibiotics. With the emergence of life-threatening drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), the need for new antibiotics is greater than ever. Yet, the development of new antibiotics has remained stagnant for nearly 50 years. A promising new class of natural products, called thiopeptides, inhibits the growth of these gram-positive bacteria at low nanomolar concentrations. Thiopeptides are exciting as potential antibiotics, because their site of action, the interface between ribosomal protein L11 and 23S rRNA, is distinct from all existing classes of antibiotics. Importantly, these natural products are derived from genetically encoded peptides, which allow for the generation of new thiopeptide analogs by simple mutagenesis. We have chosen to study the thiopeptide thiocillin as a model system, because unlike other thiopeptides, thiocillin is produced in the genetically tractable Bacillus cereus strain and is known to tolerate a variety of mutations. The immediate goal of the proposed project is to devise a method that will rapidly diversify and screen these thiopeptide natural products and thus accelerate discovery of new antibiotics. To achieve this goal, we will test the hypothesis that mutagenesis of thiocillin will provide a vast landscape of structural and chemical diversity capable of targeting antibiotic-resistant bacteria. The R21 phase of this project will focus on developing novel recombinant methods to rapidly generate large libraries of thiocillin natural products and to screen for new antibiotics. The R33 phase will develop a method to target potential resistance mutations and take promising hits through the drug development pipeline. The drug development phase will focus on increasing the solubility of thiocillin to develop an IV formulation for the treatment of systemic bacterial infections. If successful, the proposed project will have a significant impact on public health because the development of new antibiotic drugs is necessary to combat the rising problem of emerging drug resistance. Moreover, it will lay the groundwork for generating a new diverse class of natural products to screen for many other therapeutic applications.